Abstract: The transcription factor HNF4a is known to be expressed throughout digestive epithelia in humans. Two classes of HNF4a isoforms have been identified, P1 and P2. The P2 isoform class has an upregulated expression in the context of several carcinomas of the digestive system, namely in the liver and colon. The elucidation of novel protein interactors of P2 HNF4a could help to identify new biological roles for this isoform class and could also clarify HNF4a's role in the cancerous phenotypes of the colon.
Using isotopic labelling by a SILAC-based quantitative proteomics approach, I sought to identify proteins associated with the P2-HNF4a interactome. Furthermore, I used gene ontology enrichment analyses to associate identified interactors to a specific biological function. Following these findings, I supplemented the investigations with other approaches in order to substantiate suspected novel roles for the factor. Classical western blotting confirmed HNF4a's presence in DNA damage response complexes. Furthermore, HNF4a was shown to colocalize with DNA double strand break foci following genotoxic injury. Finally, double strand break reporter systems identified that P2-HNF4a decreases non-homologous end joining (NHEJ) efficiency but has no effect on homologous recombination (HR). These results support for the first time that HNF4a could participate in the DNA damage response in normal and colorectal cancer cells by interacting with the machinery specifically recruited to sites of DNA damage.
Discovery of a DNA repair role for HNF4a could represent a previously unknown biological role for P2 HNF4a. Moreover, this role in DNA damage response would be the first non-transcriptional role associated with the protein. These findings could present researchers with new targets for therapeutic remedies in colorectal cancers.